Colony stimulating factors (CSF) are produced by T-cells, macrophages, fibroblasts, and endothelial cells, and these cells are widely distributed throughout the body. The known CSFs include GM-CSF, M-CSF, and G-CSF. Among them, GM-CSF is a granulocyte macrophage-colony stimulating factor, and acts on stem cells of granulocytes or macrophages to induce their proliferation and differentiation, thereby stimulating colony formation of granulocytes or macrophages. M-CSF (macrophage-CSF) is a macrophage-colony stimulating factor, and primarily functions to stimulate colony formation of macrophages. G-CSF (granulocyte-CSF) is a granulocyte-colony stimulating factor, and stimulates colony formation of granulocytes and induces the final differentiation.
Conventionally, in order to isolate and purify G-CSF, cells are cultured and G-CSF proteins are isolated from the culture supernatant. However, this method has a problem of the low yield of G-CSF, and thus is not suitable for mass-production. In addition, Chugai Pharmaceuticals Co., Ltd. (Japan) has developed a method of producing glycosylated hG-CSF in a mammalian cell by employing a genomic DNA or cDNA including a polynucleotide encoding hG-CSF (Korean Patent NOS. 47178, 53723 and 57582). However, it is known that the sugar chain of glycosylated hG-CSF is not necessary for the activity of hG-CSF and the production of glycosylated hG-CSF employing mammalian cells requires expensive materials and facilities, and therefore, such a process is not economically feasible.
There have been attempts to produce non-glycosylated hG-CSF by employing a prokaryotic cell. In these studies, hG-CSFs having a methionine residue attached at the N-terminus thereof due to the ATG initiation codon are produced, but this form is different from the native form. Further, hG-CSF produced in a microorganism may be contaminated with impurities derived from host cells or culture materials, and a complicated purification process is required for application to high-purity medicine. Furthermore, when E. coli is used as a host cell, most of the hG-CSFs are deposited in the cells as insoluble inclusion bodies, and they must be converted to an active form through a refolding process, at a significant loss of yield. During the process, partial reduction, intramolecular disulfide formation or erroneous disulfide formation is induced, and thus a cumbersome process is needed to remove them and loss of potency is caused. One cysteine residue does not participate in forming the disulfide bond, and thus exist in a free form, resulting in additional loss of potency and reduction of stability in a protein solution.
Accordingly, there is a need to develop a method for mass-producing hG-CSFs that have no methionine residue at their N-terminus and thus are identical to the native form even though using microorganisms.
In order to solve the problems, the present inventors have previously reported that a new secretory signal peptide with high expression rate is prepared by modifying the known signal peptide of E. coli thermoresistant enterotoxin II (Korean Patent No. 316347) and used to produce native hG-CSF. Further, the present inventors have prepared an expression vector including a recombinant gene that is prepared by linking the hG-CSF gene, instead of enterotoxin gene, next to the modified signal peptide of E. coli thermoresistant enterotoxin II, and they have transformed E. coli with the expression vector, thereby expressing biologically active hG-CSFs in the periplasm by employing a microbial secretory system (Korean Patent No. 356140).
By using the microbial system of secreting a protein into the periplasm, native hG-CSFs having no methionine residue at the N-terminus can be obtained in a soluble form. Further, the periplasmic proteins are normally less than 10% of the total cell protein and thus, so less extensive purification of the recombinant protein is required than for proteins located in the cytoplasm. Furthermore, a procedure of cell disruption is not needed, and contamination with saccharides and nucleic acids present in the cytoplasm can be minimized. However, because of low expression level in the periplasmic production, its industrialization is difficult. Therefore, there is an urgent need to develop an efficient method for purifying expressed proteins with high yield and purity.